Question:

The transient receptor potential channel TRPV1 is known to be gated by noxious heat. Until now most investigations of molecular mechanisms for heat transduction were performed using ramp-shaped heat stimuli by perfusion with heated extra cellular solutions whereas the gold standard in human experiments is laser stimulation reaching noxious temperatures within milliseconds. We therefore used a laser heat stimulator for investigating laser-heat responses.

Method:

An infrared diode-laser (wavelength 1470nm, max. output 10W) was coupled into an inverted microscope (Olympus IX-81 equipped with CellR-System) via a scanning system (UGA-40; Rapp OptoElectronic, Hamburg, Germany) and focussed to a spot diameter of 50µm. Calcium transients in response to laser stimuli were investigated in HEK293 cells heterologously expressing TRPV1 with the fluorescent calcium sensor FURA-2.

Results:

Of 62 non-transfected HEK293-cells, none displayed significant changes in intracellular calcium ([Ca²⁺]_i) in response to laser pulses of up to 70mJ (7W, 10ms) or to capsaicin (10µM). In contrast, two thirds of cells transfected with TRPV1 responded to laser stimulation of 3W for =2ms (=6mJ) with a significant increase in [Ca²⁺]_i =23%. The vast majority of those (146/154) responded to both, laser-pulses (5ms; by 92.6±92.5%) and capsaicin (515±404%). Of the remaining, 8 cells responded to laser stimuli but not capsaicin, 11 to capsaicin but not heat. Heat responses had a rise time of 2.2±1.1s (10-90%) and a width at half maximum amplitude of 9.4±4.9s (n=120), and increased with stimulus intensity. When applied to hairy skin of human volunteers diode laser stimuli of 37mJ (370ms, 100mW) induced heat pain (numeric rating scale 19.8±15.6 of 100) and laser-evoked potentials with a typical N2P2 component (Ampl. 18.1± 1.7µV, N2-Latency 296±17ms; n=8).

Conclusion:

These findings confirm that heat transduction mechanisms by TRPV1 can be induced within a few milliseconds using near infrared laser stimuli.